Download electrical activity in the radial nerve cord and ampullae of sea urchins

J. Exp. Biol. (1965), 43, 247-256
With 6 text-figures
Printed in Great Britain
247
ELECTRICAL ACTIVITY IN THE RADIAL NERVE CORD
AND AMPULLAE OF SEA URCHINS*
BY D. C. SANDEMANf
Department of Zoology, University of California, Los Angeles
(Received 8 February 1965)
INTRODUCTION
Electrical activity in the radial nerve cord of the sea urchin Diadema setosum has
recently been reported by Takahashi (1964), who recorded nerve impulses following
direct photic stimulation of the isolated nerve cord. No other electrical activity has
been recorded from sea-urchin nervous systems. The following is a report on electrical
phenomena in the isolated radial nerve cord and ampullae of sea urchins, induced by
electrical stimulation.
MATERIAL AND METHODS
The experiments were performed mainly on Strongylocentrotus franciscanus in Los
Angeles; similar phenomena were observed in Tripneustes sp. and Toxopneustes sp.
studied in Lower California, near La Paz. The test sizes of the experimental animals
ranged from 7 to 15 cm. in diameter.
The radial nerve cord was stripped out of the test after cutting its lateral branches
and removing the overlying radial water-vascular canal. The isolated cord was then
supported across recording and stimulating wick electrodes. To record from an
ampulla, a portion of the test bearing the radial nerve cord and the water-vascular canal
was separated from the animal. A lobe of an ampulla was supported on one recording
electrode (the second recording electrode was grounded with the preparation), and a
short length of the radial cord, freed from the test, was placed on the stimulating
electrodes. Recordings from the side branches of the nerve cord were made using
similar relatively intact preparations. The lateral branches of the nerve cord were cut
peripherally and dissected away from the test; one was then lifted out of the grounded
solution covering the radial nerve cord, and placed over stimulating or recording
electrodes.
The stimulus was provided by a Grass SD 5 stimulator and the electrical impulses
from the nerve cord were recorded with conventional AC-coupled amplifiers.
RESULTS
(1) The radial nerve cord
A single shock produces a complex potential in the nerve cord after a latency which
is proportional to the distance between the stimulating and recording electrodes. The
• Aided by grants to T. H. Bullock from the National Institute of Health, National Science Foundation and Office of Naval Research.
f Permanent address: Gatty Marine Laboratory and Department of Natural History, University of
St Andrews, Scotland.
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D. C. SANDEMAN
conduction velocity of the initial deflexion, measured with two recording electrodes
along the length of the nerve cord, is between 14 and 20 cm./sec. and the duration
of the entire potential produced by maximal stimulus is of the order of 200 msec.
Stimulus shocks are more effective if applied with the cathode proximal to the recording
site. The reverse polarity causes a longer latency and a decrease in the amplitude of
the response. Crushing the tissue between the stimulating and recording electrodes
abolishes the response.
Alteration of the intensity or duration of the single stimulus shock alters the amplitude of the response (Fig. 1). Two main components of the complex potential can be
recognized if the stimulus intensity or duration is gradually increased from a subthreshold to a maximal value. The amplitude of the first component increases evenly
and reaches its maximum when a 2 V., 2 msec, stimulus pulse is applied. At this point,
200 msec.
Fig. 1. The graded properties of the response. Superimposed oscillograph traces show the
stepped change in the amplitude of the response produced by a stepped alteration of the
intensity and duration of the stimulus. In A the intensity was changed from 1 to 8 V., the
duration being 2 msec. In B the duration of the shock ranged from 2 to 16 msec., the intensity
being maintained at 1 V. The complex nature of the potential is apparent in both A and B.
The records also suggest an equivalence of effect in the increase of the stimulus intensity and
duration within the ranges tested.
marked by an irregularity in the curve of Fig. 2, the second and slower component
appears and soon masks the smaller initial potential. The second component increases
evenly from this point with the increase in the intensity of the stimulus until its
maximum amplitude is reached. The area beneath the potentials may be regarded
as a measure of their size though it does not distinguish between the first and second
components and may exaggerate the irregularity of the transition in Fig. 2.
Decremental spread of the potential is revealed by two recording electrodes placed
at different distances from the point of stimulation. Potentials lose amplitude over a
few millimetres and no response can be recorded 5 or 6 cm. from the stimulating
electrodes. The nature of the decrement was determined by sampling the isolated
cord with four pairs of recording electrodes placed at 5 mm. intervals (Fig. 3). The
decay of the potential is almost purely logarithmic, falling to half amplitude in 7 mm.
Potentials travelling in the oral-aboral direction show the same loss of amplitude as
those travelling in the aboral-oral direction.
Pairs or bursts of stimuli produce no facilitation of the amplitude or distance of
Radial nerve cord and ampullae of sea urchins
249
8-
/
6
/
s
8
8
/
4
2
0,
i
i
i
Stimulus intensity (V.)
Fig. 2. The development of the two components of the potential. The area under the potential
was taken as a measure of its size and has been plotted on the ordinate against the intensity of
stimulation on the abscissa. The duration of the stimulus was maintained at 2 msec. The size
of the first component increases evenly to point A. It then becomes masked by the large second
component, the development of which extends smoothly from fltoC. The irregularity of the
transition between the first and second components (A to B) is probably exaggerated by the
method of estimating the size of the response.
600 >(V.
•400 msec.
Fig. 3. Decremental spread of the potential. Recordings from four places along the isolated
nerve cord. The top trace shows the potential recorded by the electrode furthest from the
stimulus. The two parts of the potential become separated as they progress along the cord,
indicating the difference in their conduction velocities. The more distal electrodes record
irregularities in the potential not apparent in the potential recorded near the stimulus. Stimulus:
single shock of 8 V. and 2 msec, duration.
D. C. SANDEMAN
250
spread of the wave. A relative refractory period of 400 msec, follows each wave.
During this time stimulus shocks evoke potentials ranging from zero amplitude
immediately after the initial wave to full amplitude after 400 msec.
The nerve cord can be split lengthwise and each half of the nerve cord responds
to stimulus intensities of the same order as the whole cord. The same pattern of
decremental spread of the potential prevails (Fig. 4). Sometimes the amplitude of
the potential from half the cord was greater and more irregular than that of the whole
30
20-
10-
I s
a
I 4
10
15
Distance from stimulus (mm.)
20
Fig. 4. Decrements] spread of the potential. The normalized height of the potential is plotted
on the ordinate against the distance of spread on the abscissa. Each point represents the average
of nine preparations and the amount of scatter is indicated. Values obtained from half the cord
are no different from those of the whole cord. The linearity of this plot indicates the almost
perfect logarithmic decay of the potential.
cord, due possibly to the relatively smaller tissue shunt between the recording electrodes. In the whole cord the potential form recorded at a proximal electrode is often
smooth and shows little complexity, whereas the potential at a distal electrode is
usually irregular, and the first and second stages of the response become more widely
separated. The two components of the response could not be separated by splitting
the cord. Transverse cuts in the nerve cord show that the entire recordable response
is confined to one of the several tracts visible in unfixed preparations of the isolated
cord. If the active tract alone is sectioned no response can be evoked from the cord.
Radial nerve cord and ampullae of sea urchins
251
Mechanical and photic stimulation of the spines and tube feet of relatively intact
preparations produce no recordable electrical discharge in the radial nerve cord. No
potential change was recorded during direct photic stimulation of the isolated nerve
cord of either the shadow-sensitive or non-shadow-sensitive species of sea urchin.
Collision of the potentials was brought about by stimulating both ends of the cord
simultaneously. The recording electrodes were placed in the centre. The stimuli were
timed so that the effects of the collision could be observed at the recording electrode
(Fig. 5). Partial occlusion of the one wave by the other was observed. There was no
greater occlusion of the potentials from the aboral pole by those from the oral pole or
vice versa. In one experiment the nerve cord was transected through half its width in
two places, the cuts being made from opposite sides so that both longitudinal halves
of the cord would be interrupted. The preparation was arranged so that the cuts lay
on opposite sides of the recording electrodes. Impulses initiated at either end of the
cord now pass each other and the central recording electrode without losing amplitude
80011S.
200 msec
Fig. 5. The collision of the potentials travelling in opposite directions in the isolated nerve cord.
A, the potential initiated at the oral end of the preparation and recorded in the centre. B, The
potential initiated at the aboral end. C, Simultaneous stimulation of both ends of the preparation
timed so that the collision occurs at the recording electrodes. The amplitude of the potential is
diminished but not entirely occluded. Stimulus: single shock of maximal intensity.
by occlusion, suggesting that the waves of excitation are confined to their respective
longitudinal halves of the cord. Occlusion here would have indicated a lateral spread
of the potentials.
Central connexions between the adjacent lateral branches of the nerve cord can
be shown if the central end of a peripherally cut lateral branch is stimulated. A recordable potential is evoked only in the neighbouring ipsilateral branches. Peripheral
pathways connecting adjacent lateral branches of the same side could not be shown,
nor central connexions across the cord between lateral branches. However, a stimulus
applied to the whole cord elicits activity in all the lateral branches on both sides and
similarly a stimulus to any lateral branch produces a small potential in the whole cord.
(2) The ampullae
Discrete contractions of the ampullae are produced by a single shock to the oral or
aboral end of the radial nerve cord of relatively intact preparations. The ampullae
nearest the stimulating electrodes contract first but those further from the stimulus
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D. C. SANDEMAN
source contract with the same apparent intensity. Latencies are of the order of i sec.
Up to four or five separate contractions of one ampulla can occur after a single
shock to the nerve cord, each contraction in the series lasting for approximately 6 sec.
and separated from the subsequent contraction by a relaxation period of i or 2 sees.
A short burst of shocks which are of subthreshold intensity if applied singly,
causes the ampulla to contract. Facilitation lasts for about 5 sec, during which time a
single, previously subthreshold, shock causes contraction.
Stimulation of half of a partially split nerve cord produces contractions of the
ampullae on both sides of the cord beyond the split. This is unchanged by cutting
•WO/^v.
8 sec.
400 MW.
•4 sec.
2 sec.
Fig. 6. Action potentials from the lobe of an ampulla. A, Activity before the application of the
stimulus, and (lower trace) after a single maximal stimulus to the radial nerve cord. T h e first
two contractions of a series are shown, separated by a period of relaxation. B, Facilitation of the
ampulla contraction. T h e first trace shows the result of applying a single subthreshold shock to
the radial nerve cord. A burst of shocks (lower trace) of the same intensity and duration produces the contraction. Stimulus: 3 V., 1 msec, duration. C, Simultaneous recordings from the
radial nerve cord (upper trace) and the ampulla (lower trace). T h e electrode recording from
the ampulla was approximately 5 mm. from the stimulus site and the electrode on the nerve
cord 15 mm. away from the stimulus. The amplification of the upper trace is less than that of
the lower trace. Stimulus: a single maximal shock.
Radial nerve cord and ampullae of sea urchins
253
between the rows of tube feet on the outside of the test to destroy any peripheral
connexions. Similarly, stimulation of the central end of a lateral branch causes contractions of the ampullae on both sides of the cord and not only those innervated by
the adjacent lateral branches. This contradicts the results obtained with electrical
recording, and shows a spread of the effect of stimulation beyond the region of
recordable potentials. Contractions of the ampullae normally brought about by electrical stimulation of the cord are prevented by the removal of the whole, or a portion
of, the associated tube feet. However, these ampullae will still contract if mechanically
stimulated.
A burst of electrical activity can be recorded from the lobe of an ampulla during
its contraction (Fig. 6). The duration of the burst corresponds precisely with the
contractions of the ampulla. The potentials within the burst are of various amplitudes
and are difficult to resolve into units; however, single small deflexions occur irregularly
during the periods of relaxation when no movement of the ampulla is apparent. These
single waves have a duration of approximately 100 msec, and vary in amplitude from
70 to 95 /iV. The correspondence between ampulla contraction and the electrical
discharge demonstrates in Fig. 6B the facilitating effect of a burst of shocks which
are subthreshold if applied singly. Simultaneous records from the ampulla and radial
nerve cord (Fig. 6 C) show the latency for the ampulla contraction to be very much
longer than that of the potentials in the nerve cord (i.e. 1 sec. compared with 30 msec).
Artifacts deliberately caused by relatively violent movements of the electrode supporting the ampulla lobe were characteristically different and small. Stimulation of
the nerve cord of intact animals will cause movement of the lantern and occasionally
synchronized contractions of the ampullae in a neighbouring ambulacrum. The
latencies for these reactions are of the order of 1 sec.
DISCUSSION
The responses in the radial nerve cord described here are different from those
reported byTakahashi (1964) whose recordings with micro-electrodes show discrete
nerve spikes of an all-or-none kind. The potentials caused by electrical stimulation
are compound, graded and long-lasting.
Two main possibilities as to the origin of the responses caused by electrical stimulation may be examined. The first, that they are due to muscular activity, is tentatively
excluded because not even microscopically observable contraction accompanied the
most vigorous stimulation of the nerve cord. Besides, there is no anatomical evidence
of muscular tissue associated with the nerve cord in sea urchins (Laverack, personal
communication). The second, that the activity is the summed electrical potential
change of a number of small nerve fibres, is supported by some evidence. Movements
of the ampullae and lantern follow electrical stimulation of the nerve cord after a
respectable latency. These contractions must be the result of activity initiated in the
nerve cord by the single shock and transmitted to the site of action as nerve impulses.
Anatomically the cord is known to consist of large numbers of fibres of less than
iji in diameter (Hamann, 1887, Laverack, 1965). The primarily complex potential
is made more uneven by thinning down the nerve cord or recording from a greater
distance from the site of stimulation. These results are consistent with the hypothesis
254
D. C. SANDEMAN
that the potential is caused by nervous activity, for in both cases the number of active
fibres from which the recordings are being taken is less and thus less smoothing
should occur.
The presence of two major components in the complex potential in the nerve cord
is suggestive of conducting elements of two kinds having different latencies, conduction
velocities and thresholds. Attempts to separate the components by splitting the nerve
cord were unsuccessful and revealed only that both components must be confined to
a single 'tract'. It may be assumed that the two types of element are equally distributed throughout this tract. Attempts to stain this and other tracts histologically
have so far been unsuccessful.
Decremental spread of the potential along the nerve cord attests to the absence of
a through-conducting system. The decrement is the same in both directions, and it is
concluded that the numbers of fibres in the portion of nerve cord from which the
recordings were taken did not decrease considerably towards the aboral pole, in spite
of the many side branches extending from the cord. The collision experiments also
support this view.
The distance over which the potential spreads in the isolated nerve cord is unchanged
by applying multiple shocks. This absence of facilitation in the nerve cord contrasts
with the clear facilitation of the ampulla contractions. Facilitation may be taking
place within the cord but in relatively few fibres, in which case the effect would not be
recorded. The logarithmic decay of the potential suggests a purely electrotonic spread
of the excitation along the nerve but the marked increase in latency with distance of
spread indicates the contrary. More information on the electrical activity of single
units within the cord is needed before predictions can be made about the nature of
possible synaptic junctions and the transmission of nervous excitation in the cord.
The collision experiments, in showing partial occlusion of one deflexion by another
from the opposite end of the preparation, suggest the existence of pathways capable of
carrying information in both directions along the same route. Separate pathways either
not extending the full length, or incapable of conducting impulses in both directions,
could explain the incomplete occlusion at the collision point. A cord consisting of
uniformly staggered short fibres would fit the experimental results.
Central or peripheral pathways connecting the lateral branches of opposite sides of
the radial nerve cord could not be demonstrated by electrical stimulation and recording.
That central transverse connexions do exist, however, is clearly shown by the contractions of ampullae following electrical stimulation of the contralateral side branches.
The failure of the electrical recording technique to show these connexions may be
due to the nervous pathways being too fine to conduct enough impulses to give a
recordable response.
The potentials recorded from the ampullae are deemed to be muscle action potentials.
They are slow (ioo msec.) and, when recorded singly, are discrete potentials. They are
always accompanied by contractions of the ampulla, whether the contractions are
brought about by electrical or locally applied mechanical stimuli. The possibility that
these impulses are from sensory cells in the walls of the ampulla, signalling deformation
of the tissue, may be excluded because gentle stretching of the ampulla produces no
discharge. Comparison of ampulla contraction with the potential in the nerve cord
caused by the same shock reveals a large difference in their latencies. The relation
Radial nerve cord and ampullae of sea urchins
255
between the two phenomena is difficult to establish, for the repetitive electrical discharge associated with the ampulla contractions is long-lasting and shows cyclic activity
after all traces of recordable electrical activity have ceased in the radial nerve cord. The
potential in the cord may trigger a local reaction which can be detected only in the
region of the cord adjacent to the active ampulla. The ampulla contractions are
undoubtedly caused by nervous excitation in the cord, but whether this excitation
is represented by the complex potential cannot be definitely shown.
The ampulla contractions normally produced by stimulation of the nerve cord are
prevented by damaging the associated tube foot but the contractile ability of the
ampulla is not affected, as shown by its immediate response to a locally applied
mechanical stimulus. This may suggest that the necessary nervous pathways to the
ampulla run from the nerve cord via the tube foot, and the illustrations of Hamann
(1887) would support this view. However, in the starfish (Smith, 1946) a three-neuron
arc links the sensory-motor system of the tube foot, ampulla and central nervous
system. If a neural loop of this kind exists in the sea urchin, removal of the tube foot
would destroy the nervous continuity of the system without necessarily interrupting
the pathways from the radial cord to the ampulla. Hydraulically the tube foot-ampulla
system of the starfish is described as a closed system (Smith, 1946) and contraction of
the ampulla results in the extension of the tube foot. A valve between the ampulla
and the radial canal prevents fluid from flowing out of the tube foot-ampulla complex
back into the radial canal. The pressure of the fluid in the radial canals is not considered to contribute significantly to the actual extension of the tube foot (Smith,
1946). In sea urchins a slightly different situation exists. Extension of the tube foot
is not accompanied by one large contraction of the ampulla but by a number of smaller
contractions, separated by periods of relaxation. This pattern of contraction is the
same as that induced by electrical stimulation of the cord. It is inferred that the
ampullae are pumping water from the radial canals into the tube feet during their
extension. In such a system it is likely that neural feedback loops link the tube foot
and its ampulla, signalling the relative distension of the different parts of the system.
Removal of the tube foot may therefore not interfere directly with any neural pathway
at all, but the immediate change in hydraulic pressure due to the removal of the tube
foot could induce a block to the normal incoming commands from the central nervous
system. This type of control would have functional significance to an animal in which
the tube foot was damaged, for continued attempts to extend the damaged foot would
cause an unnecessary loss of fluid from the water-vascular system. The prompt contraction of the 'inhibited' ampulla to locally applied mechanical stimulation can
probably be explained by reflex activity, involving only the nerve and muscle cells of
the ampulla.
The nervous systems of the echinoderms have remained little understood in spite
of substantial study (Smith, 1965). The whole phylum presents the same peculiar
problems for the neurophysiologist in that the nervous elements are very small and
apparently hard to show histologically. However, good results have been obtained
with the electron microscope and the presence of recordable electrical activity in the
nervous system is encouraging. The animals also afford good preparations requiring
little or no care with regard to perfusion, and parts of the animal appear to behave in
much the same way as the whole animal. The electrical recording technique reported
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D. C. SANDEMAN
in this paper is not as satisfactory as could be desired in that behavioural reactions
are not always correlated with electrical potentials. Refinements of the technique are
undoubtedly possible. The preparation which may prove to be the most interesting
is that of the tube foot and ampulla, for here some anatomical details are known, at
least for the asteroids.
SUMMARY
1. A single shock applied through wick electrodes to the isolated radial nerve cord
of a sea urchin produces a recordable potential in the cord. The potential is conducted
along the cord at a velocity of between 14 and 20 cm./sec.
2. The potential is complex and graded. Two components of the potential can be
identified and have different thresholds to stimulation, conduction velocities and
amplitudes. They are believed to represent two classes of fibres.
3. The potential is conducted decrementally along the cord and normally cannot
be recorded at distances greater than 60 mm. from the stimulus. The amplitude of
the potential decays logarithmically falling to half after 7 mm. spread. There is no
facilitation of amplitude or distance of spread.
4. Potentials initiated simultaneously at either end of the isolated nerve cord collide
and partially occlude each other.
5. Stimulation of a side branch of the nerve cord evokes potentials recordable from
only ipsilateral neighbouring side branches and the whole cord. However, contractions
of the contralateral ampullae following stimulation of lateral branches reveal spread of
the excitation beyond the region of recordable potentials.
6. A single shock to a cord still attached to the test causes contraction of the associated ampullae. One ampulla will contract several times after a single shock, a period
of relaxation following each contraction.
7. Electrical activity recorded from the ampullae, and lasting many seconds after
the single shock, corresponds with their contractions. The activity is believed to be
muscle action potentials.
8. Evidence of a feedback from damaged tube feet to the cord, suppressing ampulla
response to cord stimulation, was found.
The author would like to thank Dr T. H. Bullock for his help in providing research
facilities at the University of California, Los Angeles, and also for his criticism of the
manuscript.
REFERENCES
HAMANN, 0.(1887). Beitrage zur Histologie der EMnodermen. III. Anatomie undHistologie der Echiniden
und Spatangiden. Jena: Fischer.
LAVERACK, M. S. (1965). Fine structure of the radial nerve cord of Echinus esculentus. (unpublished).
SMITH, J. E. (1946). The mechanism and innervation of the star-fish tube foot-ampulla system. Phil.
Tram. B, 333, 279-310.
SMITH, J. E. (1965). Echinodermata. In Structure and Function in the Nervous Systems of Invertebrates,
T. H. Bullock and G. A. Horridge. San Francisco: W. H. Freeman and Company.
TAKAHASHI, K. (1964). Electrical responses to light stimuli in the isolated radial nerve cord of the sea
urchin Diadema setosum (Leske). Nature, Land., 201, 1343-14.